Project Summary/Abstract Chronic exposure to tobacco smoke accounts for ~6 million deaths per year making health complications from smoking the primary cause of preventable mortality in the world. Current smoking cessation aids including nicotine replacement therapy and medication that target nicotinic receptors are minimally effective. Thus, a better understanding of brain regions, neural circuits, and neurotransmitter systems that underlie nicotine withdrawal syndrome is needed to facilitate the development of new therapies for smoking cessation. While dopaminergic (DAergic) neurons in the ventral tegmental area (VTA) that project to the nucleus accumbens are critical for nicotine reward and reinforcement, recent studies have identified the habenulo-interpeduncular (Hb- IPN) axis as a critical circuit in nicotine withdrawal symptoms. In particular, the IPN is implicated in increased anxiety during nicotine withdrawal in rodents and receives inputs from cholinergic/glutamatergic medial habenula neurons associated with affective behavior. Interestingly, we have identified a novel circuit consisting of VTA neurons that project to the IPN, the mesointerpeduncular pathway. Our data indicate that DA from VTA afferents may innervate the IPN to modulate neuronal activity and anxiety-like behavior via DA receptor signaling. In addition, DA receptor expression in the IPN appears to be regulated by chronic nicotine exposure. These observations provide the foundation for this application. We propose to test the hypothesis that VTA DAergic neurons that project to the IPN modulate anxiety and are critical for increased anxiety during nicotine withdrawal. In Aim 1, we propose to use molecular and biophysical approaches to test the hypothesis that DA controls IPN neuronal activity through activation of DA receptors that are regulated by nicotine. In Aim 2, we will use pharmacology, optogenetics, and biophysical approaches to test the hypothesis that the IPN receives DA input from the VTA. Finally, in Aim 3 we will use pharmacology, optogenetics, and behavior to test the hypothesis that the mesointerpeduncular circuit modulates anxiety-like behavior at baseline and during nicotine withdrawal. It is anticipated that understanding the role of the mesointerpeduncular circuit in anxiety and nicotine withdrawal will provide novel strategies to alleviate anxiety and nicotine withdrawal symptoms.